The human immunodeficiency virus type 1 (HIV) is the etiological agent of AIDS. HIV infection is spread through the virus's ability to package its genome into a ~120 nm protein capsid, surround it with a lipid bilayer, and transmit infectious particles to neighboring cells, tissues and other persons. HIV assembly is driven by Gag polyprotein precursor that alone can assemble and release virus-like particles carrying a dimeric RNA genome. Critical progress in the field has provided us with structural snapshots of individual domains of HIV Gag, structural models of the RNA genome and its packaging signal, the structure of the hexameric capsid lattice, as well as images of intact immature and mature HIV virions. Live cell imaging using fluorescently tagged Gag has allowed direct visualization of HIV assembly in living cells. Yet despite this progress, we face an intellectual gap between available structural snapshots of individual conformations, and the dynamic nature of the assembly process. Here we propose to establish fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET) imaging to monitor the conformational changes of the capsid and the genome during HIV assembly. Towards this end, we have already established the technology needed to site-specifically label recombinant Gag and genomic RNA molecules with Cy3 and Cy5 fluorophores, which will permit smFRET imaging. Fluorescently labeled Gag and genomic RNA molecules, in the presence of excess unlabeled material, assemble into HIV particles of the correct size in an in vitro assembly reaction that depends on the additional presence of phosphoinositols. With these tools in hand, we will monitor the conformation of a single Gag molecule during the assembly into a viral particle. We have similarly labeled the HIV packaging signal to monitor the conformational dynamics of HIV RNA genomes as they are packaged as dimers into growing HIV particles. A detailed knowledge of the energy landscape and the kinetics of HIV assembly will aid in the identification of novel structural intermediates. This new information will be relevant for the rational design of antiviral therapies. PUBLIC HEALTH RELEVANCE: This application proposes to establish fluorescence correlation spectroscopy (FCS) and single- molecule fluorescence resonance energy transfer (smFRET) imaging to monitor the conformational changes of the capsid and the genome during HIV assembly. A detailed knowledge of the energy landscape and the kinetics of HIV assembly will aid in the identification of novel structural intermediates. This new information will be relevant for the rational design of antiviral therapies.